In prior efforts, we have characterized the crystal structure of the CD4-pMHCI] complex and demonstrated that HIV-1 gp120 binds to the identical structural elements of CD4 used by pMHCII as well as to additional interaction sites. This bolstered affinity usurps normal CD4-pMHCII binding necessary for helper T cell function, thereby resulting in immunodeficiency. To establish protective immunity by vaccination, immunogens that elicit both T cell responses and broadly crossneutralizing antibody responses are essential. The present proposal has three aims. First, we will carry out rational subunit design predicated on structural analysis of trimeric SIV Mac32H envelope proteins and derivative "mini-protein" fragments. Both eukaryotic (Lec3.2.8.1 and Pichia pastoris) and prokaryotic (E. coli) expression systems will be used for protein generation. Resulting structures will be validated by NMR, X-ray crystallography and/or mAb reactivity profiling by surface plasmon resonance. The ability of envelope mini-proteins alone or linked to "molecular" adjuvants (such as the protein G B1 domain, C3d or Blys proteins) to generate neutralizing antibodies against various isolates will be examined in mammals. Second, comparable production of ADA HIV-1 gp140 envelope or mini-proteins will be tested against eight other CCR5 subtype primary isolates or dual tropic B clade isolates. Furthermore, the ability to crossneutralize isolates of C, A and O clades will be assessed and additional relevant mini-proteins devised as needed. Third, in conjunction with Project 3, we will functionally test all of the identified organic inhibitors of the CD4-HIV gpl40 protein-protein interaction for their capacity to block HIV viral infection in vitro in the absence of attendant immunosuppressive activity.